Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680

Summary Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.     

Mutations in the rfbT Gene Are Responsible for the Ogawa to Inaba Serotype Conversion in Vibrio cholerae O1

In cultures of Vibrio cholerae strains of Ogawa serotype, variant strains which had undergone serotype conversion from Ogawa to Inaba were identified. The rfbT genes cloned from the parent strains were found to produce a 31-kDa protein in the maxicell system, and to cause serotype conversion when introduced into E. coli cells expressing Inaba serotype specificity. On the other hand, rfbT genes cloned from the variant strains neither produced the 31-kDa protein nor caused serotype conversion. Nucleotide sequence of these rfbT genes as well as those of two clinical Vibrio cholerae strains of Inaba serotype revealed that mutations causing premature termination of their rfbT genes were invariably present in strains expressing Inaba serotype specificity. The result strongly suggested that genetic alteration of the rfbT gene is responsible for serotype conversion of Vibrio cholerae O1.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from cholera outbreaks in north India

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR’s revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent 0129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Variable gene family usage of protective and non‐protective anti‐Vibrio cholerae O1 LPS antibody heavy chains

Vibrio cholerae causes cholera, an enteric disease of humans that is a worldwide problem. The O1 serogroup of Vibrio cholerae contains two predominant serotypes (Inaba and Ogawa) of LPS, a proven protective antigen for humans and experimental animals. We generated B‐cell hybridomas from mice immunized with either: (i) two doses of purified Inaba LPS; (ii) two doses of an Inaba hexasaccharide conjugate (terminal six perosamine bound to a protein carrier), (iii) four doses of purified Inaba LPS; or (iv) a low dose of purified Inaba LPS followed by a booster with the Inaba conjugate. We showed previously that the first and third immunization protocols induce vibriocidal antibodies, as does the fourth; the second protocol induces antibodies that bind Inaba and Ogawa LPS but are not vibriocidal. Anti‐LPS mAbs derived from hybridomas resulting from each immunization protocol were characterized for binding to Inaba and Ogawa LPS, their vibriocidal or protective capacity, and the variable heavy chain family they expressed. LPS immunogens selected different LPS‐specific B cells expressing six different Vh chain families. Protective and non‐protective mAbs could express variable regions from the same family. One mAb was specific for Inaba LPS, the other mAbs were cross‐reactive with both LPS serotypes. Sequence comparison suggests that the pairing of a specific light chain, somatic mutation, or the specific VDJ recombination can modulate the protective capacity of mAbs that express a common variable heavy chain family member.     

wbeT sequence typing and IS1004 profiling of Vibrio cholerae isolates

Aims: To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. Methods and Results: Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII‐digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. Conclusions: IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. Significance and Impact of the Study: Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.     

Major Shift of Toxigenic V. cholerae O1 from Ogawa to Inaba Serotype Isolated from Clinical and Environmental Samples in Haiti

In October of 2010, an outbreak of cholera was confirmed in Haiti for the first time in more than a century. A single clone of toxigenic Vibrio cholerae O1 biotype El Tor serotype Ogawa strain was implicated as the cause. Five years after the onset of cholera, in October, 2015, we have discovered a major switch (ranging from 7 to 100%) from Ogawa serotype to Inaba serotype. Furthermore, using wbeT gene sequencing and comparative sequence analysis, we now demonstrate that, among 2013 and 2015 Inaba isolates, the wbeT gene, responsible for switching Ogawa to Inaba serotype, sustained a unique nucleotide mutation not found in isolates obtained from Haiti in 2012. Moreover, we show that, environmental Inaba isolates collected in 2015 have the identical mutations found in the 2015 clinical isolates. Our data indicate that toxigenic V. cholerae O1 serotype Ogawa can rapidly change its serotype to Inaba, and has the potential to cause disease in individuals who have acquired immunity against Ogawa serotype. Our findings highlight the importance of monitoring of toxigenic V. cholerae O1 and cholera in countries with established endemic disease.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

The characteristics of the reactogenicity and immunological activity of a new cholera bivalent chemical vaccine based on the results of controlled trials]

The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made.     

Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae

Defined chromosomal mutations that lead to assembly failure of the toxin coregulated pilus (TCP) of Vibrio cholerae provide useful insights into the biogenesis of a type-4 pilus. Mutants in rfb affecting LPS O-antigen biosynthesis, and strains depleted of the cytoplasmic membrane-associated ATP-binding protein TcpT, provide contrasting TCP export-defective phenotypes acting at different locations. Mutants in the perosamine biosynthesis pathway of V. cholerae 569B result in an rfb phenotype with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found in the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a large family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer membrane transporters in Gram-negative bacteria. The behaviour of translocation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flotation in density gradients, chemical cross-linking, and detergent extraction experiments. From the data presently available, it would appear that TcpA requires TcpT-mediated translocation from the cytoplasmic membrane and that TcpT stabilizes the subunit at or immediately beyond this stage, before crossing the outer membrane.     

Immunogenicity of a Killed Bivalent (O1 and O139) Whole Cell Oral Cholera Vaccine,Shanchol, in Haiti

Background

Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae.

Methodology/Principal Findings

We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6–17 years), and 47 younger children (1–5 years) in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine.

Conclusions/Significance

A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.     

An N-[(R)-(-)-2-Hydroxypropionyl]-α-L-Perosamine Homopolymer Constitutes the O Polysaccharide Chain of the Lipopolysaccharide from Vibrio cholerae O144 Which Has Antigenic Factor(s) in Common with V. cholerae O76

Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144). The LPS of O144 contained D -glucose, D -galactose, L -glycero-D -manno-heptose, D -fructose, D -quinovosamine (2-amino-2,6-dideoxy-D -gluco-pyranose) and L -perosamine (4-amino-4,6-dideoxy-L -manno-pyranose). The perosamine, a major component sugar of the LPS from O144, was in an L -configuration, as is also the case in the LPS from V. cholerae O76 (O76), in contrast to the D -configuration of the perosamine in the LPS of V. cholerae O1. A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an α(1 → 2)-linked homopolymer of (R)-(-)-2-hydroxypropionyl-L -perosamine. The serological cross-reactivity between O144 and O76 was clearly revealed by cross-agglutination and cross-agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red-blood cells that had been sensitized with the LPS from O144 and O76. In contrast, in passive hemolysis tests, the LPS of O144 did not cross-react serologically with the LPSs from other strains such as V. cholerae O1 (Ogawa and Inaba), V. cholerae O140, Vibrio bio-serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9. The LPSs from these strains consist of O polysaccharide chains composed of α(1 → 2)-linked homopolymers of D -perosamine with various N-acyl groups, and they share the Inaba antigen factor C of V. cholerae O1 in common. The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V. cholerae O1.     

Cloning part of the region encoding biosynthetic enzymes for surface antigen (O-antigen) of Salmonella typhimurium

Summary The rfb gene cluster of Salmonella typhimurium encodes the enzymes required for the biosynthesis of the O-Antigen. A part of it has been cloned in plasmid vectors pBR322 and pUC9 using an adjacent, previously cloned, part of the his operon (Barnes 1981) as a molecular probe for the first clone. A detailed restriction enzyme map of 7.57 kb of rfb DNA is presented and the approximate locations of two of the genes, rfbK and rfbM have been defined.     

Expression, purification and characterization of recombinant phosphomannomutase and GDP-{alpha}-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-{alpha}-D-mannose from D-mannose

The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis)of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase(EC 5.4.2.8 [EC] ) and GDP-     

Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems

The Yersinia enterocolitica O:3 lipopotysaccharide O-antigen is a homopotymer of 6-deoxy-L-altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementatton experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for 0-antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP-l -rhamnose biosynthesis. Rhamnose and 6-deoxy-l -altrose are C3-epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O-antigen exporters.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.